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The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H‐transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme–substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water‐mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme–substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme. [ABSTRACT FROM AUTHOR]

The study of enzymes never disappoints. Despite its long history—almost 150 years following the first documented use of the word enzyme in 1878—the field of enzymology advances apace. This long journey has witnessed landmark developments that have defined modern enzymology as a broad discipline, leading to improved understanding at the molecular level, as we aspire to discover the complex relationships between enzyme structures, catalytic mechanisms and biological function. How enzymes are regulated at the gene and post‐translational levels and how catalytic activity is modulated by interactions with small ligands and macromolecules, or the broader enzyme environment, are topical areas of study. Insights from such studies guide the exploitation of natural and engineered enzymes in biomedical or industrial processes; for example, in diagnostics, pharmaceuticals manufacture and processing technologies that use immobilised enzymes and enzyme reactor‐based systems. In this Focus Issue, The FEBS Journal seeks to highlight breaking science and informative reviews, as well as personal reflections, to illustrate the breadth and importance of contemporary molecular enzymology research. [ABSTRACT FROM AUTHOR]

Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X‐ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light‐activated proteins are attractive targets for time‐resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light‐absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small‐scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time‐resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light‐activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time‐resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time‐resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods. [ABSTRACT FROM AUTHOR]

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2O2‐driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped‐flow spectroscopy, alongside electron paramagnetic resonance and UV‐Vis spectroscopy, we reveal how H2O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions. [ABSTRACT FROM AUTHOR]

Nigel Scrutton FRS is Professor of Molecular Enzymology and Biophysical Chemistry at the University of Manchester and former Director of the Manchester Institute of Biotechnology (MIB). He obtained a first degree in Biochemistry from King's College London and followed this with a PhD at the University of Cambridge. His doctoral research, undertaken in Richard Perham's laboratory, yielded fundamental breakthroughs in enzyme redesign that have stood the test of time. Nigel was awarded a ScD degree by the University of Cambridge in 2003. After faculty positions at the University of Leicester, Nigel was appointed Professor at the University of Manchester in 2005. Over the last 15 years, he has cemented his reputation as a world leader in the fields of enzyme engineering and biocatalysis, synthetic biology, biophysics and biomanufacturing, notably by establishing and directing the Synthetic Biology Research Centre 'SYNBIOCHEM' and UK Future Biomanufacturing Research Hub. In recognition of his scientific contributions, he has received many academic awards and accolades, including being elected as Fellow of the Royal Society earlier this year. In this interview, he highlights how fundamental studies of enzymatic catalysis and mechanisms are driving key advances in biotechnology and biomanufacturing, and describes how the experiences and mentors of his formative years helped to shape his successful career at the interface between discovery and application‐focused science. [ABSTRACT FROM AUTHOR]

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light‐dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR–Pchlide–NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo‐electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation – a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development. [ABSTRACT FROM AUTHOR]

In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three‐step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an 'aldol switch' is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable 'aldol switch' consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue‐specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high‐resolution structural analysis of multiple enzyme homologues with classical site‐directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. Enzymes: TKS, EC 2.3.1.206; OAC, EC 4.4.1.26; chalcone synthase, EC 2.3.1.74; stilbene synthase, EC 2.3.1.95; 2‐PS, EC 2.3.1.-. Accession numbers: The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number 6GW3. [ABSTRACT FROM AUTHOR]

Most ene‐reductases belong to the Old Yellow Enzyme (OYE) family of flavin‐dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron‐withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X‐ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual‐specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a β‐hairpin motif in an active site loop of both these enzymes were studied using stopped‐flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the β‐hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15‐fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55‐fold) increase in efficiency in the reductive half‐reaction, when compared to the essentially unreactive wild‐type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs. [ABSTRACT FROM AUTHOR]

How the dynamics of proteins assist catalysis is a contemporary issue in enzymology. In particular, this holds true for membrane‐bound enzymes, where multiple structural, spectroscopic and biochemical approaches are needed to build up a comprehensive picture of how dynamics influence enzyme reaction cycles. Of note are the recent studies of cytochrome P450 reductases (CPR)–P450 (CYP) endoplasmic reticulum redox chains, showing the relationship between dynamics and electron flow through flavin and haem redox centres and the impact this has on monooxygenation chemistry. These studies have led to deeper understanding of mechanisms of electron flow, including the timing and control of electron delivery to protein‐bound cofactors needed to facilitate CYP‐catalysed reactions. Individual and multiple component systems have been used to capture biochemical behaviour and these have led to the emergence of more integrated models of catalysis. Crucially, the effects of membrane environment and composition on reaction cycle chemistry have also been probed, including effects on coenzyme binding/release, thermodynamic control of electron transfer, conformational coupling between partner proteins and vectorial versus 'off pathway' electron flow. Here, we review these studies and discuss evidence for the emergence of dynamic structural models of electron flow along human microsomal CPR–P450 redox chains. [ABSTRACT FROM AUTHOR]

The haem-containing mono-oxygenase cytochrome P450 3A4 ( CYP3A4) and its redox partner NADPH-dependent cytochrome P450 oxidoreductase ( CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane-bound in the endoplasmic reticulum ( ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady-state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7-benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono-oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4-nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR- CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR- CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity. [ABSTRACT FROM AUTHOR]

Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through an inferred dynamic cycle of 'open' and 'closed' conformations of the enzyme (PLoS Biol, 2011, e1001222). However, these studies were restricted to stopped-flow/FRET analysis of the reductive half-reaction, and were compromised by fluorescence quenching of the acceptor by the flavin cofactors. Here we have improved the design of the FRET system, by using dye pairs with near-IR fluorescence, and extended studies on human cytochrome P450 reductase to the oxidative half-reaction using a double-mixing stopped-flow assay, thereby analysing in real-time conformational dynamics throughout the complete catalytic cycle. We correlate redox changes accompanying the reaction chemistry with protein dynamic changes observed by FRET, and show that redox chemistry drives a major re-orientation of the protein domains in both the reductive and oxidative half-reactions. Our studies using the tractable (soluble) surrogate electron acceptor cytochrome c provide a framework for analysing mechanisms of electron transfer in the endoplasmic reticulum between cytochrome P450 reductase and cognate P450 enzymes. More generally, our work emphasizes the importance of protein dynamics in intra- and inter-protein electron transfer, and establishes methodology for real-time analysis of structural changes throughout the catalytic cycle of complex redox proteins. [ABSTRACT FROM AUTHOR]

The general requirement for conformational sampling in biological electron transfer reactions catalysed by multi-domain redox systems has been emphasized in recent years. Crucially, we lack insight into the extent of the conformational space explored and the nature of the energy landscapes associated with these reactions. The nitric oxide synthases ( NOS) produce the signalling molecule NO through a series of complex electron transfer reactions. There is accumulating evidence that protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the haem oxygenase domain, where NO is generated. Simple models based on static crystal structures of the isolated reductase domain have suggested a role for large-scale motions of the FMN-binding domain in shuttling electrons from the reductase domain to the oxygenase domain. However, detailed insight into the higher-order domain architecture and dynamic structural transitions in NOS enzymes during enzyme turnover is lacking. In this review, we discuss the recent advances made towards mapping the catalytic free energy landscapes of NOS enzymes through integration of both structural techniques (e.g. cryo-electron microscopy) and biophysical techniques (e.g. pulsed-electron paramagnetic resonance). The general picture that emerges from these experiments is that NOS enzymes exist in an equilibrium of conformations, comprising a 'rugged' or 'frustrated' energy landscape, with a key regulatory role for calmodulin in driving vectorial electron transfer by altering the conformational equilibrium. A detailed understanding of these landscapes may provide new opportunities for discovery of isoform-specific inhibitors that bind at the dynamic interfaces of these multi-dimensional energy landscapes. [ABSTRACT FROM AUTHOR]

The temperature dependence of kinetic isotope effects ( KIEs) has emerged as the main experimental probe of enzymatic H-transfer by quantum tunnelling. Implicit in the interpretation is a presumed role for dynamic coupling of H-transfer chemistry to the protein environment, the so-called 'promoting motions/vibrations hypothesis'. This idea remains contentious, and others have questioned the importance and/or existence of promoting motions/vibrations. New experimental methods of addressing this problem are emerging, including use of mass-modulated enzymes and time-resolved spectroscopy. The pressure dependence of KIEs has been considered as a potential probe of quantum tunnelling reactions, because semi-classical KIEs, which are defined by differences in zero-point vibrational energy, are relatively insensitive to kbar changes in pressure. Reported combined pressure and temperature ( p- T) dependence studies of H-transfer reactions are, however, limited. Here, we extend and review the available p- T studies that have utilized well-defined experimental systems in which quantum mechanical tunnelling is established. These include flavoproteins, quinoproteins, light-activated enzymes and chemical model systems. We show that there is no clear general trend between the p- T dependencies of the KIEs in these systems. Given the complex nature of p- T studies, we conclude that computational simulations using determined (e.g. X-ray) structures are also needed alongside experimental measurements of reaction rates/ KIEs to guide the interpretation of p- T effects. In providing new insight into H-transfer/environmental coupling, combined approaches that unite both atomistic understanding with experimental rate measurements will require careful evaluation on a case-by-case basis. Although individually informative, we conclude that p- T studies do not provide the more generalized insight that has come from studies of the temperature dependence of KIEs. [ABSTRACT FROM AUTHOR]

How cobalamin-dependent enzymes promote C-Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5′-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C-Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12-binding Rossmann domain to achieve a catalytically competent 'closed' conformational state. In 'closed' conformations of OAM, Glu338 is thought to facilitate C-Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C-Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C-Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5′-deoxyadenosyl group of B12 potentiate C-Co bond homolysis in 'closed' conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of 'closed' conformations of OAM. In 'closed' conformations, Glu338 interacts with the 5′-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C-Co homolysis, and is a crucial component of the approximately 1012 rate enhancement achieved by cobalamin-dependent enzymes for C-Co bond homolysis. [ABSTRACT FROM AUTHOR]

The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide ( NO) and carbon monoxide ( CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue ( R124) and a distal leucine residue ( L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124 A variant. In the R124 A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16 A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124 A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range. [ABSTRACT FROM AUTHOR]

The classical picture of enzyme catalysis relies on controlling the entropic and enthalpic contributions by manipulating reaction barriers and co-locating reactants and cofactors to facilitate the reaction chemistry. Catalysis is linked inextricably to the geometry of the enzyme-substrate complex and the chemical/physical properties of the active site, and probably to dynamical contributions that guide reactants along the desired reaction coordinate. Coenzyme B12-dependent enzymes have remarkable catalytic power and unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. Here we discuss recent developments that are beginning to provide atomistic insight into how coenzyme B12-dependent enzymes steer reactants along the reaction coordinate. Such insight will ultimately generate 'movies' of the catalytic process across all relevant time scales. In the longer term, this will enable more predictive engineering of this class of enzyme to achieve new and desirable chemical outcomes. [ABSTRACT FROM AUTHOR]

Enzymes are natural catalysts, controlling reactions with typically high stereospecificity and enantiospecificity in substrate selection and/or product formation. This makes them useful in the synthesis of industrially relevant compounds, particularly where highly enantiopure products are required. The flavoprotein pentaerythritol tetranitrate ( PETN) reductase is a member of the Old Yellow Enzyme family, and catalyses the asymmetric reduction of β-alkyl-β-arylnitroalkenes. Under aerobic conditions, it additionally undergoes futile cycles of NAD( P) H reduction of flavin, followed by reoxidation by oxygen, which generates the reactive oxygen species ( ROS) hydrogen peroxide and superoxide. Prior studies have shown that not all reactions catalysed by PETN reductase yield enantiopure products, such as the reduction of ( E)-2-phenyl-1-nitroprop-1-ene ( PNE) to produce ( S)-2-phenyl-1-nitropropane ( PNA) with variable enantiomeric excess ( ee). Recent independent studies of ( E)- PNE reduction by PETN reductase showed that the major product formed could be switched to ( R)- PNA, depending on the reaction conditions. We investigated this phenomenon, and found that the presence of oxygen and ROS influenced the overall product enantiopurity. Anaerobic reactions produced consistently higher nitroalkane ( S)- PNA product yields than aerobic reactions (64% versus 28%). The presence of oxygen dramatically increased the preference for ( R)- PNA formation (up to 52% ee). Conversely, the presence of the ROS superoxide and hydrogen peroxide switched the preference to ( S)- PNA product formation. Given that oxygen has no role in the natural catalytic cycle, these findings demonstrate a remarkable ability to manipulate product enantiopurity of this enzyme-catalysed reaction by simple manipulation of reaction conditions. Potential mechanisms of this unusual behaviour are discussed. [ABSTRACT FROM AUTHOR]

At least half of all enzyme-catalysed reactions are thought to involve a hydrogen transfer. In the last 10 years, it has become apparent that many of these reactions will occur, in part, or in full, by quantum mechanical tunnelling. We are particularly interested in the role of promoting vibrations on H transfer, and the Old Yellow Enzyme family of flavoproteins has proven to be an excellent model system with which to examine such reactions. In this minireview, we describe new and established experimental methods used to study H-tunnelling in these enzymes and we consider some practical issues important to such studies. The application of these methods has provided strong evidence linking protein dynamics and H-tunnelling in biological systems. [ABSTRACT FROM AUTHOR]

Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary aminesl-proline,l-pipecolic acid and sarcosine, with optimal catalytic activity towardsl-proline. The holoenzyme contains one FAD, FMN and ATP per αβ complex, is not reduced by sulfite, and reoxidizes slowly following reduction, which is typical of flavoprotein dehydrogenases. Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction withl-proline, for which a bell-shaped dependence (p Ka values 7.0 ± 0.2 and 7.6 ± 0.2) for kcat/ Km as a function of pH was observed. The pH dependence of kcat is sigmoidal, described by a single macroscopic p Ka of 7.7 ± 0.1, tentatively attributed to ionization ofl-proline in the Michaelis complex. The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis. Studies with H225A, H225Q and Y251F mutants ruled out participation of these residues in a carbanion-type mechanism. The midpoint potential of enzyme-bound FAD has a linear temperature dependence (− 3.1 ± 0.05 mV·C°−1), and extrapolation to physiologic growth temperature for P. furiosus (100 °C) yields a value of − 407 ± 5 mV for the two-electron reduction of enzyme-bound FAD. These studies provide the first detailed account of the kinetic/redox properties of this hyperthermophilicl-proline dehydrogenase. Implications for its mechanism of action are discussed. [ABSTRACT FROM AUTHOR]

Human methionine synthase (hMS) is a multidomain cobalamin-dependent enzyme that catalyses the conversion of homocysteine to methionine by methyl group transfer. We report here the 1.6 Å crystal structure of the C-terminal activation domain of hMS. The structure is C-shaped with the core comprising mixed α and β regions, dominated by a twisted antiparallel β sheet with a β-meander region. These features, including the positions of the active-site residues, are similar to the activation domain of Escherichia coli cobalamin-dependent MS (MetH). Structural and solution studies suggest a small proportion of hMS activation domain exists in a dimeric form, which contrasts with the monomeric form of the E. coli homologue. Fluorescence studies show that human activation domain interacts with the FMN-binding domain of human methionine synthase reductase (hMSR). This interaction is enhanced in the presence of S-adenosyl-methionine. Binding of the D963E/K1071N mutant activation domain to the FMN domain of MSR is weaker than with wild-type activation domain. This suggests that one or both of the residues D963 and K1071 are important in partner binding. Key differences in the sequences and structures of hMS and MetH activation domains are recognized and include a major reorientation of an extended 310-containing loop in the human protein. This structural alteration might reflect differences in their respective reactivation complexes and/or potential for dimer formation. The reported structure is a component of the multidomain hMS : MSR complex, and represents an important step in understanding the impact of clinical mutations and polymorphisms in this key electron transfer complex. [ABSTRACT FROM AUTHOR]

Photoreceptors control cellular processes in response to light. Most photoreceptors sense blue or red light, but the recent discovery of the cobalamin‐dependent photoreceptor, CarH, has expanded the wavelength range of photoreception to other regions of the electromagnetic spectrum to include the green light region. Further identification of cobalamin‐dependent green light‐sensitive photoreceptors has been hampered owing to poor annotation of the light responsiveness of cobalamin‐binding domains (CBDs) in public databases. Here we report a computational workflow, SignatureFinder, that uses a combination of sequence and structural analyses to identify new light‐responsive CBD‐containing proteins. The light response of exemplar proteins containing the proposed signature were confirmed experimentally. A structural analysis of these new photoreceptors, including the crystal structure of a new CBD domain, highlights how the signature elements interact with the cobalamin chromophore to sense light. Database mining of 128 000 CBD‐containing sequences using the identified signature revealed more diverse CBD‐containing photoreceptors, thereby expanding the family of green‐light photoreceptors. A SignatureFinder web server is available (https://enzymeevolver.com) for wider applications, including the identification of signature sequences of other biological ligands of interest. [ABSTRACT FROM AUTHOR]

This FEBS Journal Special Issue showcases the latest findings in the field of Flavins and Flavoproteins. Two reviews summarize ultrafast fluorescence techniques for studying flavoenezymes and protein dynamics of nitric oxide synthase. Research articles include studies of oxidases, oxygenase and dehydrogenase, electron transfer flavoprotein, photoreceptors, enzyme inhibition, flavoenzyme for bioconversion, regulation of riboflavin biosynthesis, and the use of physical principles for interpreting protein dynamics. [ABSTRACT FROM AUTHOR]

Electron transferring flavoproteins (ETFs) are soluble heterodimeric FAD-containing proteins that function primarily as soluble electron carriers between various flavoprotein dehydrogenases. ETF is positioned at a key metabolic branch point, responsible for transferring electrons from up to 10 primary dehydrogenases to the membrane-bound respiratory chain. Clinical mutations of ETF result in the often fatal disease glutaric aciduria type II. Structural and biophysical studies of ETF in complex with partner proteins have shown that ETF partitions the functions of partner binding and electron transfer between (a) a 'recognition loop', which acts as a static anchor at the ETF-partner interface, and (b) a highly mobile redox-active FAD domain. Together, this enables the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. This 'conformational sampling' enables ETF to recognize structurally distinct partners, whilst also maintaining a degree of specificity. Complex formation triggers mobility of the FAD domain, an 'induced disorder' mechanism contrasting with the more generally accepted models of protein-protein interaction by induced fit mechanisms. We discuss the implications of the highly dynamic nature of ETFs in biological interprotein electron transfer. ETF complexes point to mechanisms of electron transfer in which 'dynamics drive function', a feature that is probably widespread in biology given the modular assembly and flexible nature of biological electron transfer systems.

Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary amines L-proline, L-pipecolic acid and sarcosine, with optimal catalytic activity towards L-proline. The holoenzyme contains one FAD, FMN and ATP per alphabeta complex, is not reduced by sulfite, and reoxidizes slowly following reduction, which is typical of flavoprotein dehydrogenases. Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction with L-proline, for which a bell-shaped dependence (pK(a) values 7.0 +/- 0.2 and 7.6 +/- 0.2) for k(cat)/K(m) as a function of pH was observed. The pH dependence of k(cat) is sigmoidal, described by a single macroscopic pK(a) of 7.7 +/- 0.1, tentatively attributed to ionization of L-proline in the Michaelis complex. The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis. Studies with H225A, H225Q and Y251F mutants ruled out participation of these residues in a carbanion-type mechanism. The midpoint potential of enzyme-bound FAD has a linear temperature dependence (- 3.1 +/- 0.05 mV x C degrees (-1)), and extrapolation to physiologic growth temperature for P. furiosus (100 degrees C) yields a value of - 407 +/- 5 mV for the two-electron reduction of enzyme-bound FAD. These studies provide the first detailed account of the kinetic/redox properties of this hyperthermophilic L-proline dehydrogenase. Implications for its mechanism of action are discussed.

The roles of His181, His184 and Tyr186 in PETN reductase have been examined by mutagenesis, spectroscopic and stopped-flow kinetics, and by determination of crystallographic structures for the Y186F PETN reductase and reduced wild-type enzyme-progesterone complex. Residues His181 and His184 are important in the binding of coenzyme, steroids, nitroaromatic ligands and the substrate 2-cyclohexen-1-one. The H181A and H184A enzymes retain activity in reductive and oxidative half-reactions, and thus do not play an essential role in catalysis. Ligand binding and catalysis is not substantially impaired in Y186F PETN reductase, which contrasts with data for the equivalent mutation (Y196F) in Old Yellow Enzyme. The structure of Y186F PETN reductase is identical to wild-type enzyme, with the obvious exception of the mutation. We show in PETN reductase that Tyr186 is not a key proton donor in the reduction of alpha/beta unsaturated carbonyl compounds. The structure of two electron-reduced PETN reductase bound to the inhibitor progesterone mimics the catalytic enzyme-steroid substrate complex and is similar to the structure of the oxidized enzyme-inhibitor complex. The reactive C1-C2 unsaturated bond of the steroid is inappropriately orientated with the flavin N5 atom for hydride transfer. With steroid substrates, the productive conformation is achieved by orientating the steroid through flipping by 180 degrees , consistent with known geometries for hydride transfer in flavoenzymes. Our data highlight mechanistic differences between Old Yellow Enzyme and PETN reductase and indicate that catalysis requires a metastable enzyme-steroid complex and not the most stable complex observed in crystallographic studies.